Kiyoshi Mizuuchi, Ph.D.

Senior Investigator

Research Topics

Current Research

The objective of our research is to gain insights into the mechanics of cellular processes that impact the genomic structure and the heritance of the genomic material. We study mechanisms of reactions that impact the stability of the linear organization of the genome, as well as the 3-dimensional dynamics involved in the heritance of bacterial chromosomes.

Genome rearrangement by transpositional recombination

The genomes of all organisms are under the threat of assault by transposable elements. We study the mechanisms of DNA transposition of the bacteriophage Mu as a model system for a wide family of DNA rearrangement reactions from bacteria to humans. These reactions are involved in many processes that impact our health, from the spread of drug resistance among pathogenic bacteria to replication of retroviruses such as HIV. The immunoglobulin gene rearrangement reaction in vertebrates also takes place by a closely related mechanism. We use biochemical, molecular biological, and biophysical approaches, including single-molecule techniques, for our studies. We have made advances in our understanding of the reaction steps and the chemical mechanisms involved in these reactions. Our recent efforts center around the understanding of the assembly and disassembly dynamics of the macromolecular complexes involved in these reactions by developing and utilizing single-molecule biochemical approaches.

Chromosome segregation and cell division in bacteria

After DNA replication, two daughter copies of the bacterial chromosome and low copy number plasmids must be segregated into two daughter cells to ensure inheritance. Therefore, systems have evolved to actively partition the replicated copies of the genome to two halves of the cell before cell division takes place. One class of such systems involves three components: a specific DNA sequence on the segregating chromosome that functions as the bacterial equivalent of a “centromere,” a protein factor that binds to the “centromere,” and a second protein factor—an ATPase with ATP-dependent nonspecific DNA-binding activity. E. coli P1-plasmid and F-plasmid are equipped with such systems. We have reconstituted cell-free reaction systems to study these plasmid DNA partition reactions in a flow cell under a microscope. Based on the experimental results we obtained by combining biophysical and biochemical approaches, we have uncovered a previously unappreciated biological cargo transport and positioning mechanism that involves a variation of the reaction-diffusion principle, which we call diffusion-ratchet mechanism.

After segregation of replicated chromosome and plasmid copies to two halves of the cell, cell division normally must take place at the mid-cell. We study the mechanism of mid-cell localization of the cell division septum in E. coli, which is controlled by a set of Min-proteins, with biophysical techniques making use of a reconstituted cell-free reaction system. Progress in these projects is expected to advance our understanding of a variety of biomolecular tAfter segregation of replicated chromosome and plasmid copies to two halves of the cell, cell division normally must take place at the mid-cell. We study the mechanism of mid-cell localization of the cell division septum in E. coli, which is controlled by a set of Min-proteins, with biophysical techniques making use of a reconstituted cell-free reaction system observed by fluorescent microscopy. The position of cell division septum formation is determined by the location where a bacterial tubulin-homologue, FtsZ polymerizes on the inner membrane, and FtsZ polymerization is inhibited by membrane-localized MinC protein. MinC is brought to membrane by binding to MinD, an ATP-dependent membrane binding protein. Together with MinE, a stimulator of MinD ATPase, Min proteins self-organizes forming standing wave-type oscillation on the membrane inside a cell, generating time-averaged concentration minimum of MinD and MinC at mid-cell where cell division takes place. We have successfully recapitulated the standing-wave-type oscillatory dynamic pattern self-organization by MinD/E proteins in a cell-free reaction. Based on our findings, we now have a detailed mechanistic model of the system, which will be subjected to further experimental tests and refinements. Progress in these projects is expected to advance our general understanding of a variety of biomolecular transportation and patterning reactions and also to provide a foundation for the possible development of novel antibacterial agents. ransportation and patterning reactions and also to provide a foundation for the possible development of novel antibacterial agents.

Applying our Research

Progress in these projects will advance our understanding of the movement of a variety of molecular assemblies produced inside bacterial cells and could provide a basis for developing new drugs to combat disease-carrying bacteria.